The present invention relates to agents, and methods for identifying compounds, which agents and compounds result in the increased functional activity of CF-associated mutant Cystic Fibrosis Transmembrane Conductance Regulator (CFTR). In addition, the invention relates to compositions and methods for the use thereof in treating conditions that are characterized by a decrease in function of CF-associated mutant CFTR including cystic fibrosis (CF), and other protein misfolding diseases.
Cystic Fibrosis Transmembrane Conductance Regulator, a member of the ATP-binding cassette (ABC) transporter family, is believed to regulate the chloride channel responsible for cAMP-mediated chloride secretion in epithelial cells. For reviews on cystic fibrosis we refer to Guggino and Stanton, 2006) and Rowe et al., 2005. By its chloride channel function, CFTR plays a key role in chloride secretion and water balance in epithelia throughout the body. CFTR has been identified and sequenced (Riordan et al., 1989). Defects in this gene causing diminished activity and/or expression of CFTR lead to cystic fibrosis. CF is the most common fatal genetic disease in humans affecting approximately one in every 2,500 infants born in the United States of America. In patients with CF, expression of the CF-associated gene in epithelial cells leads to reduced cellular apical chloride conductance, causing an imbalance in ion and fluid transport. It is widely believed that this leads to the abnormal mucus secretion in pancreatic ductules and in the airways that ultimately results in the pulmonary infections and epithelial cell damage typically associated with disease progression in CF. In addition to respiratory problems, CF patients typically suffer from gastrointestinal problems, and pancreatic insufficiency. Males are almost uniformly infertile and fertility is decreased in females.
Sequence analysis of the CFTR gene of CF chromosomes has revealed a variety of disease-causing mutations. At present, more than 1000 mutations in the CF gene have been identified (http://www.genet.sickkids.on.ca/cftr/ or http://www.ncbi.nlm.nih.gov/entrez/dispomim.cgi?id=602421), but population studies have indicated that the most common CF mutation, a deletion of the 3 nucleotides that encode phenylalanine at position 508 of the CFTR amino acid sequence, is associated with approximately 70% of the cases of cystic fibrosis. The mutated CFTR protein is referred to as ΔF508.
It is believed that the deletion of residue 508 in ΔF508-CFTR prevents the nascent protein from folding correctly, resulting in the inability of this mutant protein to exit the endoplasmic reticulum (ER), and traffic to the plasma membrane. As a result, insufficient amounts of the mature protein are present at the plasma membrane and chloride transport within epithelial tissues is significantly reduced (Quinton, 1990). Studies have shown, however, that ΔF508-CFTR, when presented at the plasma membrane is functional as a cAMP-responsive Cl− channel (Denning et al., 1992). Correcting ΔF508-CFTR maturation, allowing exit of ΔF508-CFTR from the ER, or enhancing the activity of ΔF508-CFTR would constitute a mode of action of a novel drug to treat CF.
In fact, the cellular phenomenon of defective ER processing of ABC transporters, or other proteins, by the ER machinery has been shown to be the underlying basis not only for CF disease, but for a wide range of other isolated and inherited diseases (Ulloa-Aguirre et al., 2004). This means that drugs found for CF treatment may also be effective in the treatment of other diseases.
No therapy currently exists that restores the function of mutant CFTR. Restoring mutant CFTR function is expected to decrease CF-associated complications, and improve quality of life and expected life-span of CF patients.
Therefore, there is a clear need for molecules that facilitate the folding, processing and/or migration of the ΔF508-CFTR to the plasma membrane, thereby increasing the density of ΔF508-CFTR in the membrane, and rescuing the function of ΔF508-CFTR (correctors). These correctors may be an inhibitory agent, particularly small molecule drug compounds or biologic drugs, which target a protein regulating the processing of ΔF508-CFTR through the ER. To enable the development of such a drug, there is a need to identify target proteins, that, when antagonized, increase the density and functional performance of ΔF508-CFTR in the plasma membrane.
An example of such a protein target is syntaxin-8 (STX8), which is involved in trafficking of vesicles and has been shown to bind to the wild-type CFTR (Antonin et al., 2000; Bilan et al., 2004; Thoreau et al., 1999). It has been shown that syntaxin-8 can function as a drug target by correcting CF-associated mutant CFTR function (Fischer et al., 2006). Another positive control is BCAP31 (Lambert et al., 2001). It has been previously demonstrated that down-regulation of BCAP31 by Ad-siRNA allows functional restoration of ΔF508-CFTR (Fischer et al., 2006).
Therefore, there remains a need to identify further targets which may be of use in the diagnosis, prevention and or treatment of disorders involving ER-associated protein misfolding and in particular diseases characterized by abnormal trafficking of a disease-associated protein. Exemplary conditions include, but are not limited to, Cystic Fibrosis, Parkinson's disease, Gaucher's disease, nephrogenic diabetes insipidus, emphysema and liver disease, Maple syrup urine disease, Fabry's disease, hypogonadotropic hypogonadism, hyperinsulinemic hypoglycemia, beta-galactosidosis, Wilson's disease, long QT syndrome and retinitis pimentosa, transthyretin-linked amyloidosis, Alzheimer's disease, prion disease, and inclusion body myositis. In particular the disease is Cystic Fibrosis. As many of the clinical symptoms (e.g. airway obstruction, chronic inflammation, mucus overproduction, enhanced cytokine production) of CF overlap with those of asthma and COPD (Chronic Obstructive Pulmonary Disease), these targets may also be of use in the diagnosis, prevention and or treatment of asthma and COPD.